Suppressors of Mutation Identify New Regulators of LpxC, Which Mediates the First Committed Step in Lipopolysaccharide Biosynthesis.

Gram-negative bacteria, such as , are characterized by an asymmetric outer membrane (OM) with lipopolysaccharide (LPS) located in the outer leaflet and phospholipids facing the inner leaflet. . recruits LPS assembly proteins LapB, LapC and LapD in concert with FtsH protease to ensure a balanced biosynthesis of LPS and phospholipids.

A New Factor LapD Is Required for the Regulation of LpxC Amounts and Lipopolysaccharide Trafficking.

Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as . Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved is unknown. Examination of proteins co-purifying with LapB identified LapD as a new partner.

Checkpoints That Regulate Balanced Biosynthesis of Lipopolysaccharide and Its Essentiality in .

The outer membrane (OM) of Gram-negative bacteria, such as , is essential for their viability. Lipopolysaccharide (LPS) constitutes the major component of OM, providing the permeability barrier, and a tight balance exists between LPS and phospholipids amounts as both of these essential components use a common metabolic precursor. Hence, checkpoints are in place, right from the regulation of the first committed step in LPS biosynthesis mediated by LpxC through its turnover by FtsH and HslUV proteases in coordination with LPS assembly factors LapB and LapC.

Molecular Basis of Essentiality of Early Critical Steps in the Lipopolysaccharide Biogenesis in K-12: Requirement of MsbA, Cardiolipin, LpxL, LpxM and GcvB.

To identify the physiological factors that limit the growth of K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IV derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ() identified two independent single-amino-acid substitutions-P50S and R310S-in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene was found to be essential for the growth of Δ, Δ, Δ, and Δ() bacteria, with a conditional lethal phenotype of Δ(), which could be overcome by suppressor mutations in MsbA.

Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease.

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of Δ revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of Δ by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated , suggesting LapB and LapC act antagonistically.

Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl Isomerases Identifies Three New PPIase Activities in That Includes the DksA Transcription Factor.

Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl isomerases (PPIs) in confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of Δ6 strains. Interestingly, viability of Δ6 bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda.

Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Isomerases and Their Collective Essentiality in .

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl isomerases (PPIs). The cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed.

Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects.

Distinguishing feature of the outer membrane (OM) of Gram-negative bacteria is its asymmetry due to the presence of lipopolysaccharide (LPS) in the outer leaflet of the OM and phospholipids in the inner leaflet. Recent studies have revealed the existence of regulatory controls that ensure a balanced biosynthesis of LPS and phospholipids, both of which are essential for bacterial viability. LPS provides the essential permeability barrier function and act as a major virulence determinant.

Small regulatory bacterial RNAs regulating the envelope stress response.

Most bacteria encode a large repertoire of RNA-based regulatory mechanisms. Recent discoveries have revealed that the expression of many genes is controlled by a plethora of base-pairing noncoding small regulatory RNAs (sRNAs), regulatory RNA-binding proteins and RNA-degrading enzymes. Some of these RNA-based regulated processes respond to stress conditions and are involved in the maintenance of cellular homeostasis.

Multiple Transcriptional Factors Regulate Transcription of the rpoE Gene in Escherichia coli under Different Growth Conditions and When the Lipopolysaccharide Biosynthesis Is Defective.

The RpoE σ factor is essential for the viability of Escherichia coli RpoE regulates extracytoplasmic functions including lipopolysaccharide (LPS) translocation and some of its non-stoichiometric modifications. Transcription of the rpoE gene is positively autoregulated by Eσ and by unknown mechanisms that control the expression of its distally located promoter(s). Mapping of 5' ends of rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to Eσ-regulated promoter.

Regulated Control of the Assembly and Diversity of LPS by Noncoding sRNAs.

The outer membrane (OM) of Gram-negative bacteria is asymmetric due to the presence of lipopolysaccharide (LPS) facing the outer leaflet of the OM and phospholipids facing the periplasmic side. LPS is essential for bacterial viability, since it provides a permeability barrier and is a major virulence determinant in pathogenic bacteria. In Escherichia coli, several steps of LPS biosynthesis and assembly are regulated by the RpoE sigma factor and stress responsive two-component systems as well as dedicated small RNAs.

Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein.

Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly.

Molecular and structural basis of inner core lipopolysaccharide alterations in Escherichia coli: incorporation of glucuronic acid and phosphoethanolamine in the heptose region.

It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose.

Molecular basis of lipopolysaccharide heterogeneity in Escherichia coli: envelope stress-responsive regulators control the incorporation of glycoforms with a third 3-deoxy-α-D-manno-oct-2-ulosonic acid and rhamnose.

Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes.

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits.

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS.

Dissection of sigma(E)-dependent cell lysis in Escherichia coli: roles of RpoE regulators RseA, RseB and periplasmic folding catalyst PpiD.

To understand the mechanism of sigma(E)-dependent cell lysis, we examined the consequences of deletion derivatives of rpoE regulators rseA, rseB and rseC on sigma(E) transcription, on levels of free versus membrane-bound sigma(E) and on OMP-biogenesis limiting factor(s) that could impact cell lysis. RT-PCR showed that individual nonpolar DeltarseA and DeltarseB increased the rpoE expression to varying extents, with pronounced induction in DeltarseA. Significantly the ratio of soluble (free) versus membrane-bound form of RpoE increased in DeltarseA, however without increase of its total amount, unraveling furthermore complexity in RpoE regulation.

Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response.

To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Delta(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 degrees C, besides showing accumulation of free lipid IV(A) precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo.

A mobile loop order-disorder transition modulates the speed of chaperonin cycling.

Molecular machines order and disorder polypeptides as they form and dissolve large intermolecular interfaces, but the biological significance of coupled ordering and binding has been established in few, if any, macromolecular systems. The ordering and binding of GroES co-chaperonin mobile loops accompany an ATP-dependent conformational change in the GroEL chaperonin that promotes client protein folding. Following ATP hydrolysis, disordering of the mobile loops accompanies co-chaperonin dissociation, reversal of the GroEL conformational change, and release of the client protein.

Phosphorylation-mediated regulation of heat shock response in Escherichia coli.

Escherichia coli has two heat shock regulons under the transcriptional control of Esigma32 and EsigmaE RNA polymerases. These polymerases control the expression of genes, the products of which are needed for correct folding of proteins in the cytoplasm and the extracytoplasm respectively. In this study, we report that mutations in a tyrosine phosphatase-encoding gene led to decreased activity of these heat shock regulons.